(Fluorometric) Screening Assay Kit Thrombin Inhibitor · 11.6 Add 40 µL of Substrate Mix into each sample and control well. 11.7 Measure fluorescence on a microplate reader at Ex/Em

Version 1 Last Updated 24 March 2015

Instructions for Use

For the rapid, sensitive and accurate screening of potential inhibitors of Thrombin.

This product is for research use only and is not intended for diagnostic use.

ab197007Thrombin Inhibitor Screening Assay Kit (Fluorometric)

Discover more at www.abcam.com 1

Table of Contents

INTRODUCTION1. BACKGROUND 22. ASSAY SUMMARY 3

GENERAL INFORMATION3. PRECAUTIONS 44. STORAGE AND STABILITY 45. MATERIALS SUPPLIED 56. MATERIALS REQUIRED, NOT SUPPLIED 57. LIMITATIONS 68. TECHNICAL HINTS 7

ASSAY PREPARATION9. REAGENT PREPARATION 810. SAMPLE PREPARATION 9

ASSAY PROCEDURE and DETECTION11. ASSAY PROCEDURE and DETECTION 10

DATA ANALYSIS12. CALCULATIONS 1213. TYPICAL DATA 13

RESOURCES14. QUICK ASSAY PROCEDURE 1515. TROUBLESHOOTING 1616. FAQ 1817. INTERFERENCES 1918. NOTES 20


INTRODUCTION

1. BACKGROUND

Thrombin Inhibitor Screening Assay Kit (fluorometric) (ab197007) utilizes the ability of thrombin to cleave a synthetic AMC-based peptide substrate to release AMC, which can be detected by measuring its fluorescence at Ex/Em = 350/450 nm. In the presence of thrombin specific inhibitors, the extent of cleavage reaction is reduced or completely abolished. The loss in the fluorescence intensity can be correlated to the amount of inhibitor present in the assay solution. The kit provides a simple and rapid method to screen potential inhibitors of thrombin.

Thrombin enzyme (Factor IIa) is an important clotting factor that controls the transformation of soluble fibrinogen to insoluble active fibrin strands. Thrombin is a serine protease that catalyzes many coagulation-related reactions. Thrombin inhibitors are used as anticoagulants to prevent arterial and venous thrombosis. Some of these inhibitors are currently in clinical use while others are in clinical development.

ThrombinThrombin Substrate-AMC Cleaved Substrate + AMC (fluorescence)

Thrombin + InhibitorThrombin Substrate-AMC Decrease in fluorescence

/No fluorescence


INTRODUCTION

2. ASSAY SUMMARY

Screening compound preparation

Preparation of controls

Thrombin and substrate solution preparation

Add reaction mix

Measure fluorescence (Ex/Em = 3540/450 nm) in a

kinetic mode at 37°C for 30 – 60 min*

*For kinetic mode detection, incubation time given in this summary is for guidance only.


GENERAL INFORMATION

3. PRECAUTIONSPlease read these instructions carefully prior to beginning the assay.All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance.

4. STORAGE AND STABILITYStore kit at -20ºC in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted.Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 5.Aliquot components in working volumes before storing at the recommended temperature. Reconstituted components are stable for 2 months.


GENERAL INFORMATION

5. MATERIALS SUPPLIED

Item AmountStorage

Condition(Before

Preparation)

StorageCondition

(After Preparation)

Thrombin Dilution buffer 1 mL -20°C -20°CThrombin Assay Buffer 15 mL -20°C -20°CThrombin Enzyme 5 µL -20°C -20°CThrombin Substrate 500 µL -20°C -20°CThrombin Inhibitor (2 mM) 10 µL -20°C -20°C

6. MATERIALS REQUIRED, NOT SUPPLIEDThese materials are not included in the kit, but will be required to successfully perform this assay:

Microcentrifuge

Pipettes and pipette tips

Fluorescent microplate reader – equipped with filter for Ex/Em = 350/450 nm

96 well plate: black plates (clear flat bottoms) for fluorometric assay

Heat block or water bath


GENERAL INFORMATION

7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic

procedures.

Do not use kit or components if it has exceeded the expiration date on the kit labels.

Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.


GENERAL INFORMATION

8. TECHNICAL HINTS This kit is sold based on number of tests. A ‘test’ simply

refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions.

Keep enzymes and heat labile components and samples on ice during the assay.

Make sure all buffers and developing solutions are at room temperature before starting the experiment.

Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions.

Avoid foaming or bubbles when mixing or reconstituting components.

Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers.

Ensure plates are properly sealed or covered during incubation steps.

Ensure complete removal of all solutions and buffers from tubes or plates during wash steps.

Make sure you have the appropriate type of plate for the detection method of choice.

Make sure the heat block/water bath and microplate reader are switched on before starting the experiment.


ASSAY PREPARATION

9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening.

9.1 Thrombin Dilution Buffer:Ready to use as supplied. Equilibrate to room temperature before use. Store at -20°C.

9.2 Thrombin Assay Buffer:Ready to use as supplied. Equilibrate to room temperature before use. Store at -20°C.

9.3 Thrombin Enzyme:Dilute provided stock with 215 µL Thrombin Dilution Buffer. Mix well by pipetting up and down. Aliquot diluted enzyme so that you have enough volume to perform the desired number of tests. Avoid repeated freeze/thaw. Store at -80°C. Keep on ice while in use.

9.4 Thrombin Substrate:Ready to use as supplied. Aliquot substrate so that you have enough volume to perform the desired number of tests. Store at -20°C. Keep on ice while in use.

9.5 Thrombin Inhibitor (PPACK dihydrochloride) (2 mM):Ready to use as supplied. Aliquot inhibitor so that you have enough volume to perform the desired number of tests. Store at -20°C. Keep on ice while in use.


ASSAY PREPARATION

10.SAMPLE PREPARATIONGeneral Sample information:

We recommend that you use fresh samples. If you cannot perform the assay at the same time, we suggest that you complete the Sample Preparation step before storing the samples. Alternatively, if that is not possible, we suggest that store the samples immediately at -80°C. When you are ready to test your samples, thaw them on ice. Be aware however that this might affect the stability of your samples and the readings can be lower than expected.

10.1 Plasma samples:Plasma samples can be tested directly by adding sample to the microplate wells.However, to find the optimal values and ensure your readings will fall within the standard values, we recommend performing several dilutions of the sample.

10.2 Screening compounds:Dissolve candidate inhibitors into proper solvent. Dilute to 10X the desired test concentration with Thrombin Assay Buffer.

NOTE: We suggest using different volumes of testing compounds if effective concentration is unknown.


ASSAY PROCEDURE and DETECTION

11.ASSAY PROCEDURE and DETECTION● Equilibrate all materials and prepared reagents to room

temperature prior to use.● It is recommended to assay all controls and samples in

duplicate.11.1 Prepare Enzyme Solution:

Mix sufficient reagents for the number of assays to be performed. Prepare a master mix of the Enzyme Mix to ensure consistency.For each well, prepare 50 µL of Thrombin Enzyme Solution.

11.2 Set up Reaction wells:Set up Sample wells (S), Enzyme Control (EC) and Inhibitor Control (IC) wells as follows:

Incubate at room temperature for 10 – 15 minutes.11.3 Add 50 µL of Thrombin Enzyme Mix (Section 11.1) into each

sample and control well.11.4 Incubate at room temperature for 10 – 15 minutes.11.5 Substrate Mix:

Prepare 40 µL of Substrate Mix for each reaction:

Component Enzyme Mix (µL)

Thrombin Assay Buffer 48Diluted Thrombin Enzyme 2

ComponentSample Well (S)

(µL)

Solvent control

(BC) (µL)

Enzyme control

(EC) (µL)

Inhibitor Control (IC)

(µL)Test inhibitor compound 10 0 0

Solvent test compound

10

Thrombin Assay Buffer 0 10 9

Thrombin Inhibitor (PPACK) 0 0 1

ASSAY PRE


ASSAY PROCEDURE and DETECTION

Component Substrate Mix (µL)

Thrombin Assay Buffer 35Thrombin Substrate 5

Mix enough reagents for the number of assays (samples and controls) to be performed. Prepare a master mix of the Substrate Mix to ensure consistency. We recommend the following calculation:X µL component x (Number samples + controls +1).

11.6 Add 40 µL of Substrate Mix into each sample and control well.

11.7 Measure fluorescence on a microplate reader at Ex/Em = 350/450 nm in a kinetic mode, every 2 – 3 minutrs, for at least 30 – 60 minutes at 37°C protected from light.

NOTE: Sample incubation time can vary depending on the Thrombin activity in samples. Longer incubation times may be required if Thrombin activity is low.We recommend measuring the RFU in kinetic mode, and choosing two time points (T1 and T2) in the linear portion of the time course to calculate the Thrombin activity.Irreversible inhibitors that inhibit the Thrombin activity completely at the tested concentration will have ΔRFU = 0 and will show 100% relative inhibition.


DATA ANALYSIS

12.CALCULATIONS For statistical reasons, we recommend each sample should be

assayed with a minimum of two replicates (duplicates).12.1 Average the duplicate reading for each sample, Inhibitor

Control and Enzyme control.12.2 Subtract the mean absorbance value of the Solvent Control

(BC) if necessary from all controls and sample readings. This is the corrected absorbance.

12.3 Plot the corrected absorbance values for each sample and control as a function of the final concentration of compound.

12.4 Calculate the slope for all samples (S), Inhibition Control and Enzyme Control (EC):

𝑆𝑙𝑜𝑝𝑒 = (𝑅𝐹𝑈2 ‒ 𝑅𝐹𝑈1𝑇2 ‒ 𝑇1 ) ∗ 𝐷

Where: RFU1 = Fluorescence value at Ex/Em = 350/450 nm at Time T1.RFU2 = Fluorescence value at Ex/Em = 350/450 nm at Time T2.T1 = Time of the first reading (A1) in minutes.T2 = Time of the second reading (A2) in minutes.D = sample dilution factor.

12.5 Calculate the % Relative inhibitions as follows

% 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐸𝐶 ‒ 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝑆𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐸𝐶 × 100


DATA ANALYSIS

13.TYPICAL DATATYPICAL STANDARD CURVE – Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.

Figure 1: Inhibition of thrombin activity by a thrombin Inhibitor (PPACK Dihydrochloride).


DATA ANALYSIS

Figure 2: Thrombin activity was measured in plasma samples in the presence and absence of Thrombin Inhibitor (PPACK Dihydrochloride). S = Substrate, I = Inhibitor, AB = Activation Buffer containing Factor Xa.


RESOURCES

14.QUICK ASSAY PROCEDURENOTE: This procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time.

Prepare enzyme, substrate, inhibitor, dilution buffer and assay buffer (aliquot if necessary); get equipment ready.

Prepare samples and dissolve test inhibitors in suitable solvent.

Prepare Enzyme Mix for all wells to be set up (50 µL/well).Component Enzyme Mix (µL)

Thrombin Assay Buffer 48Diluted Thrombin Enzyme 2

Set up plate as follows:

Incubate RT 10-15 min.

Prepare 40 µL Thrombin Substrate Mix for each well (Number samples + controls + 1).

Component Substrate Mix (µL)

Thrombin Assay Buffer 35Thrombin Substrate 5

Add 40 µL of Thrombin Substrate Mix to all wells.

Measure plate on a microplate reader at Ex/Em= 350/450 nm in a kinetic mode at 37°C for 30 – 60 min protected from light.

Component Sample Well (S) (µL)

Enzyme Control

(EC) (µL)

Inhibitor Control (IC) (µL)

Enzyme Mix 50 50 50Test Inhibitor Compound 10 0 0

Thrombin Assay Buffer 0 10 9

Thrombin Inhibitor (PPACK) 0 0 1


RESOURCES

15.TROUBLESHOOTING

Problem Cause Solution

Use of ice-cold buffer Buffers must be at room temperature

Plate read at incorrect wavelength

Check the wavelength and filter settings of instrument

Assay not

workingUse of a different 96-

well plate

Colorimetric: Clear platesFluorometric: black wells/clear

bottom plateSamples not

deproteinized (if indicated on protocol)

Use PCA precipitation protocol for deproteinization

Cells/tissue samples not homogenized

completely

Use Dounce homogenizer, increase number of strokes

Samples used after multiple free/ thaw

cycles

Aliquot and freeze samples if needed to use multiple times

Use of old or inappropriately stored

samples

Use fresh samples or store at - 80°C (after snap freeze in liquid

nitrogen) till use

Sample with erratic readings

Presence of interfering substance

in the sample

Check protocol for interfering substances; deproteinize samples

Improperly thawed components

Thaw all components completely and mix gently before use

Allowing reagents to sit for extended times

on ice

Always thaw and prepare fresh reaction mix before use

Lower/ Higher readings in samples and Standards Incorrect incubation

times or temperaturesVerify correct incubation times and temperatures in protocol


RESOURCES

Problem Cause SolutionPipetting errors in

standard or reaction mix

Avoid pipetting small volumes (< 5 µL) and prepare a master mix

whenever possibleAir bubbles formed in

wellPipette gently against the wall of

the tubes

Standard readings do not follow a linear pattern Standard stock is at

incorrect concentration

Always refer to dilutions on protocol

Measured at incorrect wavelength Check equipment and filter setting

Samples contain interfering

substances

Troubleshoot if it interferes with the kit

Unanticipated results

Sample readings above/ below the

linear range

Concentrate/ Dilute sample so it is within the linear range


RESOURCES

16.FAQ


RESOURCES

17. INTERFERENCES


RESOURCES

18.NOTES


RESOURCES


RESOURCES

RESOURCES 23

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(Fluorometric) Screening Assay Kit Thrombin Inhibitor · 11.6 Add 40 µL of Substrate Mix into each sample and control well. 11.7 Measure fluorescence on a microplate reader at Ex/Em

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